Expression and Function of the Type 3
Complement Receptor in Tissues of the
Developing Mouse
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J Immunol 1998; 160:4543-4552; ;
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References
Derralynn A. Hughes and Siamon Gordon
Expression and Function of the Type 3 Complement Receptor
in Tissues of the Developing Mouse1
Derralynn A. Hughes and Siamon Gordon2
C
ell surface receptors facilitate important interactions between macrophage (Mf)3 and their environment during
murine development (1). Cellular and soluble signals coordinate the differentiation of stem cells into monocytes, which
enter the blood to form a pool of migration competent cells and
subsequently enter every organ in the body to form heterogeneous
populations of tissue Mf (2– 4). The differentiation Ag F4/80 is
first expressed in the 10-day-old embryo on monocytes and some
stellate Mf in the yolk sac, and then on Mf populations in the
liver (day 11), spleen (day 12), and bone marrow (day 17). Monocyte infiltration and local proliferation also result in the appearance
of abundant F4/801 Mf in mesenchymal organs during this period
(5). However, it is not clear whether this temporal progression of
monocyte appearance in various organs represents serial migration
or whether each organ is seeded from common sources of stem
cells. Furthermore, while previous studies have used mAb F4/80,
which recognizes a Mf-restricted plasma membrane glycoprotein
(6), to describe the distribution of Mf during development (5), the
signals and receptors involved in the entry, migration, and exit of
monocytes within developing tissues are not known.
Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
Received for publication April 9, 1997. Accepted for publication Janary 5, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the United Kingdom Medical Research Council, the
United Kingdom Arthritis and Rheumatism Council, and a Theodore Williams Scholarship from Oxford University (to D.A.H.).
2
Address correspondence and reprint requests to Dr. Siamon Gordon, Sir William
Dunn School of Pathology, University of Oxford, South Parks Rd., Oxford, U.K. OX1
3RE.
3
Abbreviations used in this paper: Mf, macrophage; PMN, polymorphonuclear leukocyte; BP, bacteriologic plastic; PO, pathology Oxford; PLP, periodate-lysine-paraformaldehyde solution; RPM, resident peritoneal macrophages; TCP, tissue culture
plastic; EAiC3b, opsonized sheep red cells; Eb, erythroblasts; TPM, thioglycolateelicited peritoneal macrophages.
Copyright © 1998 by The American Association of Immunologists
In the adult mouse, constitutive migration of monocytes from
the bone marrow maintains most resident tissue Mf populations
(7) and continues alongside inflammatory and immunologically
driven recruitment processes in which a family of structurally and
functionally related surface glycoproteins, the leukocyte b2 integrins (LFA-1, CR3, and p150,95) (8), have been implicated (9 –
11). Each of these molecules consists of an antigenically distinct
a-chain (150 –190 kDa) noncovalently associated with a b-chain
(95 kDa) as an a1b2 dimer (reviewed in Ref. 12). Congenital inability to express the b2 chain results in an impairment of surface
expression of all three heterodimers, severe defects in leukocyte
accumulation in vivo and adherence in vitro, and an increase in
life-threatening infections (13). However, the role of these molecules in the constitutive or inflammatory migration of monocytes
during development is unknown.
Similarly, while the distribution of leukocyte integrins in the
adult is well documented (14, 15), little is known of their expression during ontogeny. In the adult, CR3 is expressed on monocytes, neutrophilic polymorphonuclear leukocytes (PMN), and NK
cells in blood and hemopoietic tissues, but not on populations of
stellate tissue Mf, except microglia in the brain, marginal zone
Mf in the spleen, and Mf in the subcapsular sinus of the lymph
node (16). This suggests that CR3 expression is widely downregulated as a consequence of constitutive entry of monocytes into
tissues and differentiation into resident Mf such as Kupffer cells
and implies a possible role for CR3 in development. In contrast,
high levels of CR3 expression are retained on Mf that have been
recruited to tissues in response to inflammatory signals.
The aim of this study, therefore, was to analyze the spatial and
temporal sequences of CR3 expression in fetal and neonatal tissues
as a first step to define its role in constitutive and inflammatory
monocyte recruitment during development. Subsequently, mAb
5C6, which blocks CR3-dependent adhesion of Mf to bacteriologic plastic (BP) and has potent effects on some forms of myelomonocytic migration in vivo (17–20), also provided a useful
tool to determine the ability of CR3 on isolated neonatal Mf to
0022-1767/98/$02.00
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Macrophage (Mf) expression of the leukocyte integrins has been implicated in their adhesion and migration in the adult. Little
is known, however, of the expression or function of these molecules during development. This study defines the spatial and
temporal sequences of expression of the type 3 complement receptor (CR3) in the developing mouse; establishes the functional
efficacy of this molecule in spreading, adhesion, and phagocytosis; and investigates its role in inflammatory and constitutive
migration. Expression of CR3 on monocytes occurred early compared to Mf-restricted glycoprotein F4/80, but expression on
stellate tissue Mf appeared later than F4/80 and was transient. Expression of CR3 on resident tissue Mf is more widespread
during development, being retained on only very specific Mf populations in the adult. Neutrophil polymorphs expressed CR3
from day 17 of gestation onward. The anti-CR3 mAb 5C6 was used to investigate the role of CR3 in adhesion, spreading, and
phagocytosis by neonatal Mf. Neonatal macrophages were found to adhere, spread, and phagocytose by CR3-dependent mechanisms, and a CR3-independent system was implicated in the spreading of neonatal Mf. The role of CR3 in migration during
development was then investigated. 5C6 had potent effects on the early stages of the migration of myelomonocytic cells to an
inflammatory stimulus in vivo. Despite efficient transplacental transfer of the Ab from pregnant mother to fetus, the process by
which monocytes generate populations of resident tissue Mf was undisrupted, indicating the existence of CR3-independent
mechanisms of monocyte migration during development. The Journal of Immunology, 1998, 160: 4543– 4552.
4544
mediate adhesion, spreading, and phagocytosis. Having then established the efficacy of neonatal CR3 in vitro, its role in the processes of constitutive and inflammatory myelomonocytic migration during development was also investigated.
Materials and Methods
Animals
Embryos and newborn mice (Pathology Oxford (PO)) were bred at the Sir
William Dunn School of Pathology (Oxford, U.K.) and maintained under
conventional laboratory conditions with free access to food and water.
Females were inspected daily for the presence of a vaginal plug, which was
designated day 0 of pregnancy. The day of birth (usually day 19 of pregnancy) was designated day 0 of neonatal life.
Antibodies
Immunocytochemical techniques
Fixation and sectioning of murine organs. Embryos from day 8 to birth
and neonatal animals up to 2 wk after birth were examined. Visceral yolk
sac, liver, spleen, bone marrow, thymus, lung, kidney, and gut were dissected from conceptuses as they became readily identifiable. Organs were
washed in PBS before freezing in OCT embedding medium (Miles,
Elkhart, IN) cooled in isopentane (BDH-Merck, Poole, U.K.) over liquid
nitrogen. Frozen sections were cut on a cryostat (Leica, Wetzlar, Germany)
at 5 mm, air-dried for 1 h, and frozen at 220°C until further use. Fresh
organs were fixed for 10 min in 2% paraformaldehyde in HEPES-buffered
isotonic saline before staining; 2 mM calcium chloride was added to the
fixative to maintain the structural conformation of the integrin.
Ag detection. Fixed sections were washed in PBS containing 0.1% (v/v)
Triton X-100 (BDH-Merck) and treated with 2% normal rabbit serum for
30 min. Sections were incubated for 90 min in hybridoma supernatant,
PBS, or isotype-matched control mAb. Endogenous peroxidase activity
was inactivated by incubation of sections with 1022 M glucose, 1023 M
NaN3, and 40 U glucose oxidase in 100 ml 0.1 M phosphate buffer for 15
min at 37°C (23). Affinity-purified, mouse-adsorbed, biotinylated second
Ab (Vector) was used at 1% for 45 min, and avidin-biotin-peroxidase complex (ABC elite, Vector) (24) was used according to the supplier’s recommendation. The presence of Ag was revealed by incubation with 0.5 mg/ml
3,39-diaminobenzidine HCl (Polysciences, Northampton, U.K.) and
0.024% H2O2 in 10 mM imidazole in PBS, pH 7.4.
Double labeling. To detect a second Ag, polyclonal Abs were incubated
on sections for 90 min followed by 1% alkaline phosphatase-conjugated
goat anti-rabbit second Ab for 60 min (Vector). The Ag was revealed by
incubation with a 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium alkaline phosphatase substrate kit (Vector), and sections were counterstained in cresyl fast violet acetate (BDH-Merck) and mounted in DPX
(BDH-Merck). Sections in which primary Ab, secondary Ab, or avidinbiotin-peroxidase complex reagent were omitted and sections treated with
irrelevant control Abs showed no staining.
In vivo detection of Ags. Pregnant mice were injected i.v. with 0.5 mg of
5C6 mAb or isotype-matched control mAb, 1C5 (which does not react with
the surface of murine leukocytes) (25), on days 14 and 17 of pregnancy.
Newborn PO mice were injected i.p. with 0.05 mg of purified rat mAb.
Offspring of the pregnant mice or the newborn mice injected directly were
subsequently fixed by perfusion with 2% periodate-lysine-paraformaldehyde solution (PLP) (26). After postfixing in the same solution for 4 h,
tissues were impregnated with 20% sucrose in 0.1 M phosphate overnight
before freezing and sectioning, as described. No further fixation of these
sections was required. Affinity-purified, mouse-adsorbed, biotinylated antirat Ab (Vector) was used to locate injected rat Abs, and polyclonal rabbit
anti F4/80 antiserum was used to define the distribution of F4/801 monocytes or Mf in these sections.
Photography. Representative black and white photographs were taken using a blue filter (Wratten Gelatin Filter no. 47, Kodak, Rochester, NY) that
intensifies the brown precipitate. Color photographs were taken using a
15-cc magenta filter.
Neonatal peritoneal Mf. Resident peritoneal Mf (RPM) were obtained
from the cavities of 3-day-old PO mice by lavage with 1 ml of PBS.
Recruited cells were similarly harvested from 3-day-old PO mice that had
been injected i.p. with 25 ml of Brewer’s complete thioglycolate broth on
the day of birth.
Adhesion to artificial substrata (18). Cells to be assayed for adhesion
were resuspended in RPMI 1640 with 10% FBS and plated at a density of
3 3 105 Mf/well in flat-bottom BP or tissue culture plastic (TCP) 96-well
plates (Flow Laboratories (Rickmansworth, U.K.) and Nunc/Life Technologies (Paisley, U.K.)). mAb (5 mg/ml) alone or in combination with divalent cation chelator (5 mM EDTA) were added to test wells and incubated
for 30 min at 4°C. After incubation for 90 min at 37°C, plates were washed
three times in PBS, and adherent cells were fixed in methanol and stained
with 40% Giemsa solution for 1 h. Plates were washed in tap water, and the
retained dye was solubilized in methanol and quantified by measuring absorbance at 450 nm in an automatic plate reader (Anthos III, Denley Instruments, Billinghurst, U.K.). In some assays cell viability was assessed at
each stage using trypan blue dye exclusion (Sigma, Poole, U.K.).
Spreading assays
Cells were resuspended in RPMI 1640 plus 10% FBS and plated at a
density of 105 Mf/chamber in multichamber slides. mAb (2 mg/ml 5C6 or
control 1C5) or chelator (5 mM EDTA) was added to test chambers and
retained throughout the experiment. Slides were washed in PBS, and adherent cells were fixed in 0.25% glutaraldehyde at 30 min or 1, 3, 6, 10.5,
or 24 h. The number of cells with a spread morphology was assessed using
phase contrast microscopy and expressed as a percentage of the total adherent cells.
Phagocytosis of opsonized red cells
Sheep erythrocytes (Becton Dickinson, Oxford, U.K.; stored in Alsever’s
solution at 4°C until used) were incubated with 30% mouse IgM anti-sheep
erythrocyte in PBS (Nordic Immunologic Laboratories, Tilburg, The Netherlands) for 45 min at 4°C, followed by fresh mouse serum for 30 min at
37°C to fix iC3b. Opsonized sheep erythrocytes (EAiC3b) were used at a
5% (v/v) suspension for 60 min to assay for rosetting at 4°C or phagocytosis at 37°C (27, 28). Binding was quantified by counting the number of
erythrocytes attached to 100 Mf. Noninternalized erythrocytes were lysed
in 0.147 M NH4Cl before counting the number of erythrocytes ingested per
100 Mf.
LPS-induced myelomonocytic recruitment
Newborn PO mice were injected intradermally with 10 ml of PBS or LPS
(0.2 mg) containing 1% Monestral blue to mark the site of the lesion and
at the same time i.p. with 0.05 mg of purified 5C6 or isotype-matched
control mAb (1C5). Animals were perfusion fixed with PLP at 1, 4, 8, 10.
5, 24, and 56 h after injection and processed as described above to reveal
injected mAb and F4/80 on recruited cells.
Results
Expression of the leukocyte integrin, CR3, during murine
development
To establish a basis for manipulation of myelomonocytic cell migration during the ontogeny of the mononuclear phagocyte system,
the expression of CR3 on monocytes, Mf, and PMN during murine development was investigated. Tissue samples were taken
throughout fetal and neonatal life and stained using a combination
of two mAbs, M1/70 and 5C6, which show differential sensitivity
to digestion of the cell surface with pronase and are therefore
believed to recognize different epitopes of CR3 (18). No staining
was observed with the irrelevant isotype-matched mAbs
CAMPATH-1G or 1C5. Organs from at least two mice from each
of three independent litters were examined at each developmental
stage, and in each case a comparison was made with consecutive
sections stained with the F4/80 polyclonal Ab.
Lymphohemopoietic organs
Liver (Fig. 1). The liver primordium develops relatively earlier in
the mouse than in man and is visible at only 9 days of gestation,
when it is composed of broad hepatic cords separated by large
sinusoids containing nucleated erythroblasts (Eb). CR31 monocytes were found in the fetal liver of whole embryos from this time
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The following rat mAbs were used as hybridoma supernatants for immunohistochemistry. F4/80 (21) recognizes a 160-kDa Ag of unknown function. M1/70 (22) and 5C6 (18) were used in combination at a final dilution
of 1/10 in PBS to recognize the CR3. Biotinylated rabbit anti-rat IgG were
obtained from Vector Laboratories (Peterborough, U.K.). Polyclonal Abs
recognizing F4/80 and sialoadhesin were prepared in our laboratory by Drs.
P. Dri and P. Crocker and were routinely used at a dilution of 1/500 for
immunohistochemistry.
ONTOGENY OF MURINE CR3
The Journal of Immunology
onward, whereas F4/801 was only detected on monocytes from
day 10 of gestation (day 10e; not shown). The density of CR31
monocytes in liver increased as hemopoiesis progressed from day
11e (Fig. 1a) and by day 13e, when the liver is well developed,
labeled monocytes could be seen in clusters around liver sinusoids
(Fig. 1b). Hemopoietic foci were found intermingled within the
hepatic cords, but unlike the stellate Mf detected by F4/80, CR31
monocytes were not associated with developing Eb. The F4/801
stellate Mf first exhibited CR3 expression on day 15 (Fig. 1c). By
day 17e, CR31 PMN were apparent and located in clusters of
erythroid and myeloid cells around a CR31 stromal Mf (Fig. 1d).
CR31 PMN and monocytes increased in number until shortly after
birth (Fig. 1, e and f ) and continued to be distributed unevenly
throughout the liver, with high numbers of cells adjacent to the
sinusoids. CR3 staining of stellate Mf was maximal around 8 days
FIGURE 2. Expression of CR31 cells in the developing spleen occurs
before the onset of local hemopoiesis. a, Day 12 fetal spleen: occasional
CR31 monocytes appear in the mesenchymal mass (arrow). b, Day 13 fetal
spleen: CR31 monocytes are distributed throughout the spleen. c, Day 15
fetal spleen is now an active site of hemopoiesis. The tissue is homogeneous in architecture and contains many CR31 monocytes. d and e, Day 16
and 18 fetal spleen: some areas destined to become white pulp (w) are free
of CR31 cells. f, Day 1 newborn spleen: small areas of white pulp (wp) are
already distinct; CR31 monocytes, PMN, and Mf are present only in the
red pulp. g, Day 4 newborn spleen: hemopoiesis is reduced, but continues
throughout adult life. h, Day 8 newborn spleen: the spleen now resembles
that of a normal adult. i, Adult spleen: stellate CR31 marginal zone Mf
(mz) delineate the white pulp (wp). The red pulp (rp) contains few CR31
mature Mf but many monocytes and PMN. Bar 5 50 mm.
after birth (Fig. 1g) even though at this stage the Mf were no
longer associated with remaining erythroid cells. Hemopoietic
clusters containing CR31 monocytes were rare by this stage, and
mature Mf accounted for most of the CR3 staining. After birth,
declining hemopoietic activity in the liver was associated with a
decreased number of CR3-staining monocytes and PMN, while
CR3 staining of mature Mf progressively decreased in intensity.
In the adult, CR3 expression on spindle-shaped sinus-lining Mf
(Kupffer cells) was much reduced or absent (Fig. 1h), in contrast
to F4/80, which is highly expressed on mature Kupffer cells in the
liver (not shown).
Spleen (Fig. 2). By 11 days of gestation, the epithelial lining of
the dorsal mesentery of the stomach is thickened and represents the
anlage of the spleen. CR31 monocytes first appeared among the
cells of this rudimentary spleen on day 12e (Fig. 2, a and b), at the
same time as F4/801 expression was first detected on splenic
monocytes. Ramified F4/801 Mf appear on day 15e and are surrounded by developing red cells, but at this stage these cells do not
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FIGURE 1. CR3 is expressed on hepatic monocytes and stellate Mf
throughout murine development. a, Day 11 fetal liver: monocytes (arrows)
are scattered among endodermal cells. b, Day 13 fetal liver: the number of
CR31 monocytes has increased and are located around the margins of the
liver sinuses (s). c, Day 15 fetal liver: hemopoietic activity has increased
and CR3 is now expressed at low levels on stellate Mf as well as on
clustered monocytes. d, Day 17 fetal liver: CR31 stellate Mf are associated with clusters of erythroid and CR31 myeloid cells (both monocytes
and PMN; c). e, Day 1 newborn liver: hemopoietic activity continues, and
CR31 Mf plasma membrane processes are still associated with hemopoietic cell clusters. f, Day 3 newborn liver: hemopoietic activity is now much
reduced and CR31 plasma membrane processes of the immature Kupffer
cells (Kc) are only occasionally in contact with hemopoietic cells. g, Day
8 newborn liver: there are many highly membranous CR31 Kc. No hemopoietic clusters. h, Adult liver: CR3 is expressed on spindle shaped sinuslining Mf. Membrane staining of the cells is much reduced compared with
that in the neonate. Bar 5 50 mm.
4545
4546
ONTOGENY OF MURINE CR3
10e, when nucleated Eb were also seen within the blood spaces. At
no stage were Mf associated with clusters of Eb.
Bone marrow. Hemopoietic stem cells and F4/801 Mf invade
the cartilaginous rudiment of bone on day 16e, resulting in the
formation of the marrow cavity. CR31 monocytes and PMN were
evident within the stroma of the bone marrow from day 17e, when
hemopoietic activity is initiated (Fig. 3b). Clusters of erythroid and
CR31 myeloid cells surround central F4/801 Mf during development and in the adult mouse, but CR3 staining was only observed on the stromal Mf of the bone marrow from day 17e until
shortly after birth and, unlike F4/80, was absent from these Mf in
the adult as previously reported (29).
Nonhemopoietic tissues
express CR3 (Fig. 2c). From day 16e (Fig. 2d) the splenic architecture becomes more complex, with distinct arteries and veins and
regions destined to become lymphoid areas, containing none of the
monocytes or numerous CR31 granulocytic cells that are then apparent in the developing red pulp (Fig. 2e). CR31 monocytes and
PMN continue to be detected in the red pulp immediately after
birth and during adult life (Fig. 2, f and g). In contrast, CR31
stellate Mf were most evident in the expanding marginal zone of
the white pulp 1 wk after birth (Fig. 2h), and in the adult could be
detected both in the marginal zone and occasionally around the
central arteriole in the white pulp (Fig. 2i). The network of stromal
Mf expressing F4/80 in the red pulp never expressed CR3.
Yolk sac. CR3 was detected on stellate Mf and monocytes within
and along vessels of the vitelline yolk sac from the ninth day of
gestation (day 9e; Fig. 3a), which is 1 day earlier than F4/80 expression has been observed on cells seen mainly in the yolk sac
mesoderm; CR3 is also expressed on more stellate Mf within
vessels than F4/80. The density of CR3 staining peaked on day
Peritoneal cells
Resident and elicited neonatal peritoneal Mf were immunoreactive for CR3 (not shown).
The immunohistochemistry outlined above established that CR3
was expressed on monocytes and tissue Mf during development,
but was gradually lost after birth from mature tissue Mf in many
regions. The pattern of CR3 expression differed from that of F4/80
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FIGURE 3. CR31 myelomonocytic cells are widely distributed in other
developing organs. a, Day 9 yolk sac: both stellate Mf (sm) and monocytes (mo) are evident throughout the yolk sac. b, Day 17 fetal bone marrow: monocytes and PMN are visible in clusters (c) around stromal Mf (s).
c, Day 14 fetal gut: CR31 Mf lie just beneath the serosa of the gut but no
staining is seen within the primitive villi (arrow). d, Day 18 fetal gut: CR31
Mf are stained within the lamina propria and villi. e, Day 14 fetal lung.
There are many CR31 cells within the mesenchyme of the developing
lung. f, Day 18 fetal lung. CR31 cells are now within the loose connective
tissues of the lung. g and h, Day 17 fetal and day 1 newborn thymus: a low
frequency of CR31 monocytes is found in the cortical region (c) of the
developing thymus. Monocytes are found only in the vessels of the corticomedullary junction shortly before and after birth. Little staining is seen
in the medulla (m). Bar 5 50 mm.
F4/801 and CR31 monocytes were seen throughout the undifferentiated tissue within which organs develop. As organogenesis
proceeds, F4/801 Mf persist to become components of loose connective tissue of differentiated organs, whereas CR3 expression is
variable depending on the organ.
Gut. CR3 was first detected on spindle-shaped Mf around the
capsule of the gut on day 11e. One day later, when the lumen
became visible, CR31 Mf were still confined to the capsule,
whereas F4/801 Mf appeared in the lamina propria at this stage.
From day 13e CR31 monocytes appeared in the lamina propria
circumferential to the developing villi, and on day 15 mature Mf
were visible within the villi. The pattern of moderate CR3 expression on the stellate Mf within the villi and strong expression on
monocytes of the lamina propria continued throughout development and in the adult (Fig. 3, c and d).
Lung. The lung anlage develops on day 9e as rudiments of larynxtrachea-bronchi. CR31 Mf, with extensive processes, were evident around the outer margins of the mesenchyme of the developing lung from day 11e, at the same stage that F4/801 cells were
observed randomly distributed within the mesenchyme. By day 13
the lungs were clearly subdivided into lobes, and by day 14 the
disappearance of mesenchyme from the lung was accompanied by
the formation of airways and vessels with which some CR31
monocytes and more spindle-shaped Mf were associated (Fig.
3e). CR31 cells, often in pairs, and F4/801 Mf were distributed
throughout the intraalveolar septa of the lung from just before birth
(Fig. 3f ), whereas the alveolar Mf populations of the adult failed
to express CR3 or F4/80.
Mf of the kidney failed to express CR3 at any point during
development, whereas F4/80 was expressed on Mf between the
tubules from day 12e.
Thymus. The thymus becomes visible as epithelial lobes surrounded by a thin outer capsule of mesenchyme at 13 days of
gestation. Flattened F4/801 cells can be seen in the thymus from
this time. As the organ grows, the F4/801 Mf increase in number
and become more stellate, the majority of Mf were found in the
cortical regions among developing thymocytes. By contrast, CR31
monocytes could only be detected in the vascularized corticomedullary region of the thymus from day 16e onward when the thymus
increases strikingly in volume and the flattened F4/801 Mf do not
express detectable levels of CR3 at any point during development
or in the adult (Fig. 3, g and h).
The Journal of Immunology
4547
Table I. Expression of F4/80 and CR3 during murine development
F4/80
CR3
Monocytes
Mf
Liver
Spleen
d10e–d7pn
d9e–d7pn
d10–14e in
mesoderm
d11e–adult
d15e–adult
d9e–14e in
vitelline vessels
d9e–d7pn
d12e–adult
Bone marrow
Gut
d17e–adult
d12e–adult
d16e–adult
d12e–adult
d17e–adult
d13e in lamina
propria-adult
Yolk sac
Lung
Thymus
d11e–adult
d13e–adult
Kidney
d12e–adult
Monocytes
d9e–14e in
mesoderm
d15e–d7pn
d7pn–adult
marginal zone
D17e–d4pn
d11e in capsule d15e
in lamina propria-adult
d11e–d7pn
PMN
d17e–d7pn
d16/17e–adult
d17e–adult
d16e–adult corticomedullary junction
No expression
sion, and phagocytosis of neonatal peritoneal Mf were assessed at
a stage when there was still significant expression of CR3 on the
stromal Mf of spleen and liver.
CR3-dependent adhesion of neonatal Mf
mAb 5C6 was initially selected for its ability to inhibit the adhesion of adult Mf to BP by blocking the interaction between CR3
and an undefined component of FBS that coats the plastic surface
(18). This interaction is also inhibited by EDTA, since the receptor
function of integrins depends on the presence of divalent cations
(30, 31). In this study, short term adhesion assays to BP were
performed with neonatal and adult peritoneal cells lavaged from
the resting peritoneal cavity or after its stimulation 3 days previously with thioglycolate broth. The comparative adhesion qualities
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both spatially and temporally (summarized in Table I). CR3 was
only retained on the marginal zone Mf of the spleen and the subcapsular Mf of the lymph nodes (not shown), which may be involved in trafficking of leukocytes into these organs in the adult.
This was suggestive of a specific role for CR3 during the developmental period and was consistent with a possible role for CR3 in
the adhesion or migration of monocytes into and within organs
before they differentiate into resident tissue Mf. It was impossible
to obtain neonatal peripheral blood monocytes in sufficient numbers to assess their use of CR3 in vitro; however, in the adult RPM
have been successfully used as surrogate monocytes in adoptive
transfer assays of migration (25). Isolated neonatal peritoneal Mf
were therefore used to establish the functional competence of the
CR3 expressed during murine development. The spreading, adhe-
FIGURE 4. CR3-dependent
adhesion
and
spreading of neonatal and adult peritoneal Mf. A
and B, Adhesion of neonatal and adult Mf to bacteriologic plastic: preincubation of the cells with
5C6 mAb or chelation of divalent cations with
EDTA inhibited the adhesion of both RPM and TPM
to BP. Each data point represents eight replicate
wells; results were confirmed in four independent
experiments. C, Spreading of neonatal and adult
peritoneal Mf on glass: 100% neonatal cells exhibited ramified morphology 21 h before the adult cells.
Each data point represents three replicate wells; results were confirmed in three independent experiments. D, The spreading of neonatal and adult peritoneal Mf at 6 h was only partially inhibited by
incubation with 5C6 mAb and was more fully inhibited by chelation of divalent cations using EDTA.
Each data point represents three replicate wells; results were confirmed in three independent
experiments.
Mf
4548
ONTOGENY OF MURINE CR3
of the cells and the effects of chelation of divalent cations or preincubation of the cells with mAb 5C6 are shown in Figure 4, A and
B. The adhesion of neonatal peritoneal Mf to BP, like that of adult
Mf, was divalent cation dependent and almost completely inhibited by 5C6. The CR3 of neonatal cells is therefore functional in
adhesion to BP. However, in contrast to the adult where thioglycolate-elicited peritoneal Mf (TPM) were more adherent than
RPM, neonatal RPM adhered more readily to BP than
neonatal TPM.
Role of CR3 in the spreading of neonatal Mf
Role of CR3 in phagocytosis by neonatal Mf
In addition to adhesion and spreading, CR3 is known to mediate
binding and ingestion of complement-opsonized particles by adult
peritoneal Mf (33, 34). Figure 5 summarizes the binding and
phagocytosis of iC3b EAiC3b by neonatal and adult resident and
recruited Mf. As previously documented (34), adult TPM were
able to both bind and ingest EAiC3b, whereas RPM bound opsonized particles well, but ingested relatively few. Similarly, in the
neonate both RPM and TPM bound EAiC3b, but ingestion of
EAiC3b was greater by TPM than by RPM. In each case EAiC3b
rosetting was inhibited by 5C6, indicating that the CR3 on both the
resident and elicited neonatal peritoneal Mf is capable of binding
iC3b and to some extent mediates ingestion of EAiC3b.
LPS-induced myelomonocytic cell recruitment to neonatal skin
Since the epitope of CR3 recognized by 5C6 and implicated in
adult myelomonocytic cell recruitment is present and active in adhesion and phagocytosis in the neonate, the ability of 5C6 to block
inflammatory recruitment of cells in the neonate was examined. It
is well documented that thioglycolate broth elicits a 10- to 20-fold
influx of myelomonocytic cells into the adult peritoneal cavity 2 to
4 days after injection. Recruitment of myelomonocytic cells to the
neonatal peritoneal cavity was examined by i.p. injection of sterile
thioglycolate broth. However, in contrast to the adult, there was no
detectable increase in the total number of cells within the peritoneal cavity over the 7 days after thioglycolate injection. Furthermore, the developmental, weight-related increase in the number of
cells in the cavity was suppressed by thioglycolate (not shown).
FIGURE 5. CR3-dependent phagocytosis of iC3b-coated sheep
erythrocytes by neonatal and adult peritoneal cells. Neonatal and adult
RPM and day 3 TPM were plated in glass multichamber slides, and the
binding and phagocytosis of EAiC3b was assessed. Each data point
represents four replicate wells; results were confirmed in three independent experiments. A, The number of Mf with bound or internalized
opsonized erythrocytes: neonatal TPM bound and internalized EAiC3b,
whereas, like adult RPM, neonatal RPM bound cells more effectively
than they internalized. B, The number of bound or internalized opsonized erythrocytes per Mf: neonatal Mf bound and internalized
EAiC3b as effectively as comparable adult Mf.
Since injection of control Ab, PBS, or 5C6 also prevented any
developmental increase in cell number in the cavity, it is likely that
any injection results in a sufficient inflammatory stimulus to induce
the resident cells to leave or die within the cavity. For this reason
an alternative model of inflammatory recruitment in neonatal mice
was employed. It has been established that an acute inflammatory
response in adult murine skin may be elicited by LPS. After intradermal injection, myelomonocytic recruitment into adult skin
occurs within 2 h (35). This model was therefore adapted for the
neonate.
One-day-old mice were injected i.p. with control Ab or 5C6 and
intradermally with PBS or LPS, including monestral blue to mark
the site of injection. Animals were fixed by PLP perfusion at various times, and the lesions were analyzed immunohistochemically.
One hour after injection, many CR3-labeled cells were found at
the lesion, but few could be identified as monocytes using polyclonal F4/80. Cell recruitment was increased at 4 h; it was maximal
8 h after injection and consisted mainly of PMN (Fig. 6, a and b).
By 24 h the total numbers of recruited cells had started to decline,
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The interaction of Mf with more complex substrata such as serum
coated glass or TCP can be described in the two phases: adhesion
and spreading. 5C6 and EDTA, which inhibit Mf adhesion to BP,
reduce the spreading of adult cells on glass and TCP, but fail to
inhibit their adhesion. The kinetics of spreading of adult and neonatal peritoneal Mf on glass and its dependence on CR3 were
assessed using a method previously described by Haynes et al.
(32). Neonatal RPM spread more rapidly than adult RPM, such
that 100% adherent neonatal cells had an extensively arborized
morphology after only 3 h of incubation compared with 24 h for
the adult RPM (Fig. 4C). Chelation of divalent cations inhibited
the spreading of neonatal RPM on TCP ( p , 0.01), whereas
blocking CR3 with 5C6 mAb up to the 6 h point did not significantly inhibit their spreading on this surface. This indicates that the
spreading of neonatal Mf involves divalent cation-dependent receptors other than CR3 or a conformation of CR3 not recognized
by 5C6 (Fig. 4D). Since the proportion of adult Mf fully spread
at the 6 h point was small (only 18.7%), the inhibitory effect of
5C6 and EDTA (20 and 58%) did not reach statistical significance.
This time point was chosen to fall on the up-slope of spreading for
both cell populations, thus potentially allowing both positive and
negative effects of the Ab to be observed. The inhibitory effect of
5C6 and EDTA on adult cells at time points that allow full spreading has been well documented (18).
The Journal of Immunology
4549
and the infiltrate became predominantly monocytic in character
(Fig. 6, c and d). The early, largely neutrophil, response was inhibited by the i.p. injection of 5C6 mAb. Figure 6, E and F, shows
the absence of F4/801 monocytes/Mf or CR31 monocytes and
PMN at the 24 h lesion. The later phase of monocytic recruitment
appeared to be at least partially 5C6 resistant (Fig. 6, e– h), with
both CR3- and F4/80-staining cells detectable in the vicinity of the
LPS injection after 56 h. This result confirms the activity of CR3
in the inflammatory recruitment of neonatal PMN and monocytes
and also suggests that, as in some models in the adult, other adhesion molecules are also involved.
Role of CR3 in the constitutive migration of myelomonocytic
cells during murine development
We next investigated whether the migration of cells expressing
CR3 during prenatal development could be affected by 5C6. It
was essential to know that the Ab could reach sites of expression if its effects on monocyte migration were to be assessed.
Animals were injected with mAb i.v. on days 14 and 17 of
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FIGURE 6. CR3 is active in the initial
stages of LPS-induced recruitment of myelomonocytic cells to the skin of neonatal
mice. Newborn PO mice were injected intradermally with PBS or LPS containing
Monestral blue to mark the site of the lesion and i.p. with purified 5C6 or isotypematched control mAb (1C5). Animals were
fixed by perfusion with PLP at various
times after injection and stained to reveal
CR3 on PMN and monocytes (a, c, e, and
g; brown) and F4/801 monocytes and Mf
by means of a polyclonal Ab (b, d, f, and h:
blue). Results were confirmed in three independent experiments. a and b, Skin 8 h
after injection of LPS intradermally and
control mAb i.p.: CR31 PMN (p), but no
F4/80-stained monocytes are visible at the
site of the lesion marked with monestral
blue (mb). c and d, Skin 24 h after injection
of LPS intradermally and control mAb i.p.:
CR31 PMN (brown) and CR31 F4/801
monocytes (m, blue) are now both present
at the site of LPS injection. e and f, Skin
24 h after injection of LPS intradermally
and 5C6 mAb i.p.: the recruitment of both
PMN and monocytes was blocked by 5C6.
g and h, Skin 56 h after injection of LPS
intradermally and 5C6 mAb i.p.: CR31 F4/
801 monocytes (mo) are again visible at
the site of LPS injection.
pregnancy, and their offspring were fixed by perfusion 1 day
after birth. 5C6 could be detected on Mf in the neonatal spleen
(Fig. 7a), lamina propria of the gut (Fig. 7b), and Kupffer cells
in the liver (Fig. 7c) and had therefore crossed the placenta.
When Ab was injected i.p. into newborn mice, followed by PLP
perfusion-fixation of the whole animal, 5C6 could be detected
in the liver, thymus, and spleen 1, 4, 6, and 9 days after injection using a rat monoclonal detection system. In control animals
that received PBS or isotype-matched control Ab, no in vivo
labeling of cells could be detected (not shown). In animals injected with 5C6, PMN, monocytes, and some tissue Mf were
strongly labeled in the developing hematolymphoid organs.
Monocytes and marginal zone Mf were labeled in the spleen
(Fig. 7d), monocytes were labeled in the corticomedullary region of the thymus (Fig. 7e), and Kupffer cells and monocytes
were labeled in the liver (Fig. 7f ). In the nonhematolymphoid
organs of gut, brain, and skin, CR31 cells were detected in
architectural arrangements similar to those of conventionally
stained specimens (not shown).
4550
ONTOGENY OF MURINE CR3
shows Kupffer cells in the liver 9 days after 5C6 administration,
illustrating the morphologic integrity of the organs after Ab injection. This pattern of staining was identical with that detected in
animals preinjected with control Ab and with that detected in native animals by Morris et al. (5). Therefore, while 5C6 exhibits the
capacity to cross the placenta and be retained on the surface of
myelomonocytic cells for several days after birth, such intervention does not interfere with the ability of the cells to find their
organ-specific locations.
Discussion
Rabbit polyclonal Abs against F4/80 and sialoadhesin were used
to assess the effect of 5C6 on other Mf populations. (The detection
system employed to locate the rabbit polyclonal Abs did not bind
to the injected rat mAb.) No morphologic disruption or interference with the expression of F4/80 or sialoadhesin was observed
over the extended time course over which the injected Ab could
still be detected. In a number of Ab-treated animals there appeared
to be an increased frequency of mitoses in the neonatal liver and
spleen and an enhanced sinusoidal location of monocytes in the
liver. Figure 7g shows pF4/80 staining in the spleen, and Figure 7h
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FIGURE 7. Transplacental transfer of 5C6 mAb labels CR3 on developing cells without disrupting their distribution. Pregnant (days 14 and 17
of gestation) and newborn mice (day 0) were injected i.v. with 5C6 mAb
or its isotype-matched control 1C5 mAb. Tissues were subsequently fixed
by perfusion, and the location of the injected mAb was determined using
an anti-rat IgG detection system. The effect of the mAb on organ morphology and the distribution of other Mf Ags was assessed using rabbit polyclonal F4/80. a, Day 4 neonatal spleen after injection of 5C6 into the
pregnant mother on days 14 and 17 of gestation: the 5C6 mAb has crossed
the placenta to label monocytes and PMN in the developing spleen. b, Day
4 neonatal gut after injection of 5C6 to the pregnant mother on days 14 and
17 of gestation: the injected 5C6 stains monocytes and Mf in the lamina
propria of the gut. c, Day 4 neonatal liver after injection of 5C6 to the
pregnant mother on days 14 and 17 of gestation. The injected 5C6 mAb
stains Kupffer cells (Kc). d, Newborn spleen, 1 day after injection: 5C6
labels monocytes and neutrophils within the red pulp (rp). Some labeled
cells show mitotic figures. e, Newborn thymus, 1 day after injection: 5C6
marks myelomonocytic cells in the corticomedullary junction (cmj). f,
Newborn liver, 1 day after injection of 5C6: Kc are stained by the injected
Ab. g, Spleen from 5C6 injected animal stained with pF4/80: F4/80 detects
stellate Mf within the red pulp (rp). No F4/801 cells are found in the white
pulp (wp). h, Liver from 5C6 injected animal, stained with pF4/80: F4/80
is readily detectable on Kc.
In this study we have used a combination of mAbs that recognize
different epitopes of CR3 (18, 36), a cell surface component involved in monocyte and neutrophil migration in the adult, to analyze its expression in the developing mouse. CR31 monocytes
and Mf were distinguished on account of their round or stellate
morphology and frequent presence of phagocytic material in Mf.
Neutrophils, which only became evident on day 16 or 17 of gestation, were small with a multilobed nucleus. Flow cytometric
analysis has previously demonstrated large populations of CR3positive myeloid cells in the fetal liver from day 11 of gestation,
increasing up to birth and decreasing from the first to third weeks
after birth (37). However, the study in question does not detect
heterogeneity of expression within individual organs or distinguish
between monocytes produced as a result of hemopoietic activity at
the height of the CR3 expression and mature resident tissue Mf.
Here we have compared the immunohistochemical distribution of
CR3 during development with that of F4/80 (Table I) to analyze
the expression of a known adhesion molecule on a population of
developing cells identified by an independent marker. The expression of CR3 on monocytes within most developing organs preceded the appearance of F4/801 monocytes or Mf, but the expression of CR3 on stellate tissue Mf appeared later than F4/80
and was transient, often being lost shortly after birth. The expression of CR3 on resident tissue Mf is therefore more widespread in
the fetus and neonate than in the adult, except on very specific
populations of adult Mf (Table II). LFA-1 was detected on monocytes and Mf at the same stage as CR3 in most organs, but was
retained during development and in the adult (not shown). This
raises interesting questions about the regulation of CR3 expression
during development, its function on monocytes and Mf in the
embryo and neonate, and the significance of its down-regulation in
the adult. It may be that its function is specific to an early window
of monocyte/Mf life cycle and is lost as a consequence of monocyte differentiation within tissues. For example, CR3 expression on
monocytes may be related to their capacity for migration and may
no longer be required once a Mf is fixed within a tissue. CR3
expression on mature Mf in the lymph node and spleen occurs in
areas of leukocyte entry into these organs (38); elimination of marginal zone Mf in the spleen reduces the accumulation of lymphocytes in this area (39), suggesting that these Mf have an active role
in the trafficking of other cells. Furthermore, adoptively transferred
RPM are first evident in the marginal zone of the spleen and subcapsular sinus of the lymph node (25), so that CR3 expressed in
these regions might be related to newly extravasated cells that have
not yet assumed a tissue phenotype.
The CR3 of Mf isolated from the peritoneal cavities of neonatal
mice was functional in in vitro assays of adhesion to BP and could
be completely inhibited by mAb 5C6 or EDTA. However, the
spreading of neonatal RPM, which was more rapid and extensive
than that of adult RPM, could not be completely inhibited by 5C6.
Integrins on leukocytes are normally inactive, in that they bind
with only very low avidity to their ligands but can be triggered
The Journal of Immunology
4551
Table II. Comparison of CR3 expression during development and in the adult
Adult Mouse
Developing Mouse
Yolk sac
Liver
Low level on Kupffer cells
Spleen
Bone marrow
Gut
Lung
Thymus
Kidney
Monocytes, PMN, MZ Mf
Monocytes and PMN
Monocytes and lamina propria Mf
No expression
Monocytes
No expression
a
Mf in mesoderm and monocytes in
vitelline vessels
Monocytes, PMN,
developing Kupffer cells
Monocytes, PMN, MZ a Mf after birth
Monocytes, PMN, and Mf
Monocytes and lamina propria Mf
Mf
Monocytes
No expression
MZ, marginal zone.
This study has demonstrated high levels of CR3 expression in
hematolymphoid and nonhematolymphoid organs during development that coincide with high levels of hemopoiesis and decline
after birth. Prominent expression during the developmental period
is compatible with a role for CR3 in hemopoietic cell migration.
CR3 expressed on isolated neonatal peritoneal Mf is able to mediate adhesion and phagocytosis, and it contributes to the initial
phase of myeloid cell recruitment to an inflammatory stimulus in
vivo. However, mAb 5C6 was unable to inhibit the largely monocytic, delayed phase of recruitment to LPS or the constitutive migration of monocytes during development. Mf migration in the
normal developing animal is therefore largely independent of CR3,
which may perform additional, undiscovered functions in tissue
modelling and myelomonocytic cell ontogeny.
Acknowledgments
We thank Mr. L. Turley for excellent technical support, and Mrs. E. Darley
for expert assistance with immunohistochemistry.
References
1. Gordon, S., V. H. Perry, S. Rabinowitz, L.-P. Chung, and H. Rosen. 1988. Plasma
membrane receptors of the mononuclear phagocyte system. J. Cell Sci.
9(Suppl.):1.
2. Dexter, T. M. 1982. Stromal cell associated haemopoiesis. J. Cell Physiol.
1(Suppl.):87.
3. Gallagher, J. T., E. Spooncer, and T. M. Dexter. 1983. Role of the cellular matrix
in haemopoiesis. J. Cell Sci. 63:155.
4. Haworth, C. 1989. Multifunctional cytokines in haemopoiesis. Blood Rev. 3:263.
5. Morris, L., C. F. Graham, and S. Gordon. 1991. Macrophages in haemopoietic
and other tissues of the developing mouse detected by the monoclonal antibody
F4/80. Development 112:517.
6. McKnight, A. J., A. J. Macfarlane, P. Dri, L. Turley, A. C. Willis, and S. Gordon.
1996. Molecular cloning of F4/80, a murine macrophage-restricted cell surface
glycoprotein with homology to the G-protein-linked transmembrane 7 hormone
receptor family. J. Biol. Chem. 271:486.
7. Van Furth, R., and Z. A. Cohn. 1968. The origin and kinetics of mononuclear
phagocytes. J. Exp. Med. 128:415.
8. Springer, T. A., D. Davignon, M. K. Ho, K. Kurzinger, E. Martz, and
F. Sanchez-Madrid. 1982. LFA-1 and Lyt-2,3 molecules associated with T lymphocyte-mediated killing; and MAC-1, and LFA-1 homologue associated with
complement receptor function. Immunol. Rev. 68:171.
9. Rosen, H., and S. Gordon. 1990. The role of the type 3 complement receptor in
the induced recruitment of myelomonocytic cells to inflammatory sites in the
mouse. Am. J. Respir. Cell Mol. Biol. 3:3.
10. Keizer, G. D., A. A. Te Velde, R. Schwarting, C. G. Figdor, and J. E. De Vries.
1987. Role of p150,95 in adhesion, migration, chemotaxis and phagocytosis of
human monocytes. Eur. J. Immunol. 17:1317.
11. Nishikawa, K., Y.-J. Guo, M. Miyasaka, T. Tamatani, A. B. Collins, M.-S. Sy,
R. T. McCluskey, and G. Andres. 1993. Antibodies to intercellular adhesion
molecule 1/lymphocyte function-associated antigen 1 prevent crescent formation
in rat autoimmune glomerulonephritis. J. Exp. Med. 177:667.
12. Hynes, R. O. 1992. Integrins: versatility, modulation, and signalling in cell adhesion. Cell 69:11.
13. Anderson, D. C., and T. A. Springer. 1987. Leukocyte adhesion deficiency: an
inherited defect in the Mac-1, LFA-1 and p150,95 glycoproteins. Annu. Rev. Med.
38:175.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
remotely by other membrane receptors or their own ligands to an
activated conformation (40, 41). Further studies are required to
establish whether the spreading of neonatal RPM is due to an activation-specific epitope of CR3 or to a different molecule. Enhanced spreading may be a reflection of recent extravasation from
the blood to form the resident population or may be due to the
presence of mediators within the newborn cavity, such as IFN-g
(42) and TNF (43) or macrophage CSF (44), which are known to
enhance the spreading of murine adult RPM in vitro.
To investigate the role of CR3 in inflammatory migration in
neonates, LPS was introduced into the skin of neonatal mice. The
initial phase of LPS-induced myelomonocytic recruitment in the
neonate was blocked by mAb 5C6. However, despite the continued
presence of 5C6-labeled Mf at the lesion, a later phase of myelomonocytic recruitment was still evident and therefore was independent of CR3. Analogous studies in the adult indicate that T
cell-dependent inflammation induced by specific antigenic challenge in sensitized mice is also biphasic and is inhibited by 5C6
only in its initial phase even though the Ab is present and functional for 4 or more days (20) and recruitment of cells to pulmonary granulomata following Calmette-Guérin bacillus infection or
to hepatic sinusoids in murine malaria are mostly independent of
CR3-mediated mechanisms (9). The present study shows that induced monocyte migration in the neonate also has CR3-dependent
and -independent components.
Administration of 5C6 to pregnant or newborn mice resulted in
labeling of the CR3 Ag in vivo and demonstrated that mAb 5C6
was able to cross the placenta. This is of interest since 5C6 is also
able to cross the blood-brain barrier (45), whereupon it induces
mitoses and apoptosis of microglia. Low levels of mitosis were
also observed in neonatal spleens and thymi after injection of this
Ab, indicating that ligation of CR3 in the periphery may also stimulate cell division, but further quantitative studies are needed to
establish whether this was enhanced relative to the background in
the present model. 5C6 failed to disrupt constitutive monocyte
migration to nonhematolymphoid organs or to affect myelomonocytic cell distribution in hematolymphoid organs. The expression
of other differentiation markers, including F4/80 and sialoadhesin,
was unaffected by the administration of 5C6 at or before birth. A
variety of Abs did not detect any significant disruption of Mf
distribution in tissues by 5C6 up to 9 days after its original administration. This is in keeping with studies indicating that migration of fluorescently labeled Mf from the blood to normal tissue in
the adult is CR3 independent (25) and that normal Mf populations
are found in CR3-deficient human patients to the extent examined
(13). The development of mice deficient in CR3 is also grossly
normal; however, details of Mf distribution during the embryonic
period of these animals have not been reported (46).
4552
31. Kirchofer, D., J. Grzesiak, and M. D. Pierschbacher. 1991. Calcium as a potential
physiologic regulator of integrin-mediated cell adhesion. J. Biol. Chem. 266:
4471.
32. Haynes, D. R., M. W. Whitehouse, and B. Vernon-Roberts. 1991. The effects of
some anti-arthritic drugs on the shape and function of rodent macrophages. Int.
J. Exp. Pathol. 72:9.
33. Griffin, F. M., C. Bianco, and S. C. Silverstein. 1975. Characterisation of the
macrophage receptor for complement and demonstration of its functional independence from the receptor for the Fc portion of immunoglobulin G. J. Exp. Med.
141:1269.
34. Bianco, C., F. Griffin, and S. Silverstein. 1975. Studies of the macrophage complement receptor: alteration of the receptor function on macrophage activation.
J. Exp. Med. 141:1278.
35. Andersson, P. B., V. H. Perry, and S. Gordon. 1992. The acute inflammatory
response to lipopolysaccharide in CNS parenchyma differs from that in other
body tissues. Neuroscience 48:169.
36. Beller, D. I., T. A. Springer, and R. D. Schreiber. 1982. Anti-Mac-1 selectively
inhibits the mouse and human type three complement receptor. J. Exp. Med.
156:1000.
37. Velardi, A., and M. D. Cooper. 1984. An immunofluorescence analysis of the
ontogeny of myeloid and B lineage cells in mouse haemopoietic tissues. J. Immunol. 133:672.
38. van den Berg, T. K., J. J. Breve, J. G. Damoiseaux, E. A. Dopp, S. Kelm,
P. R. Crocker, C. D. Dijkstra, and G. Kraal. 1992. Sialoadhesin on macrophages:
its identification as a lymphocyte adhesion molecule. J. Exp. Med. 176:647.
39. Kraal, G., H. Rodrigues, K. Hoeben, and N. Van Rooijen. 1989. Lymphocyte
migration in the spleen: the effect of Mf elimination. Immunology 68:227.
40. Cabanas, C., and N. Hogg. 1993. Ligand intercellular adhesion molecule 1 has a
necessary role in activation of integrin lymphocyte function-associated molecule
1. Proc. Natl. Acad. Sci. USA 90:5838.
41. Ginsberg, M. H., X. Du, and E. F. Plow. 1992. Inside-out integrin signalling.
Curr. Opin. Cell Biol. 4:766.
42. Schultz, R. M. 1980. Macrophage activation by interferons. In Lymphokine Reports. E. Pick, ed. Academic Press, New York, p. 113.
43. Beutler, B., U. Trackeno, I. Milsark, N. Krochin, and A. Cerami. 1986. Effect of
g-interferon on cachectin expression by mononuclear phagocytes: reversal of
lps(d) (endotoxin resistance) phenotype. J. Exp. Med. 164:1791.
44. Boocock, C. A., G. E. Jones, E. R. Stanley, and J. W. 1989. Colony stimulating
factor-1 induces rapid behavioural responses in the murine macrophage cell line
BAC1.2F5. J. Cell Sci. 93:447.
45. Reid, D. M., V. H. Perry, P.-B. Andersson, and S. Gordon. 1993. Mitosis and
apoptosis of microglia in vivo induced by an anti-CR3 antibody which crosses the
blood brain barrier. Neuroscience 56:529.
46. Coxon, A., P. Rieu, F. J. Barkalow, S. Askari, A. H. Sharpe., U. H. von Adrian,
M. A. Arnaout, and T. N. Mayadas. 1996. A novel role for the b2 integrin
CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation.
Immunity 5:653.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
14. Flotte, T. J., T. A. Springer, and J. Thorbecke. 1983. Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidermal sheets.
Am. J. Pathol. 11:112.
15. Larson, R. S., and T. A. Springer. 1990. Structure and function of leukocyte
integrins. Immunol. Rev. 114:181.
16. Gordon, S., L. Lawson, S. Rabinowitz, P. R. Crocker, L. Morris, and V. H. Perry.
1992. Antigen markers of macrophage differentiation in murine tissues. Curr.
Top. Microbiol. Immunol. 181:1.
17. Hutchings, P., H. Rosen, L. O’Reilly, E. Simpson, S. Gordon, and A. Cooke.
1990. Transfer of diabetes in mice prevented by blockade of adhesion-promoting
receptor on macrophages. Nature 348:639.
18. Rosen, H., and S. Gordon. 1987. Monoclonal antibody to the murine type 3
complement receptor inhibits adhesion of myelomonocytic cells in vitro and inflammatory cell recruitment in vivo. J. Exp. Med. 166:1685.
19. Rosen, H., S. Gordon, and R. J. North. 1989. Exacerbation of murine listeriosis
by a monoclonal antibody specific for the type 3 complement receptor of myelomonocytic cells. J. Exp. Med. 170:27.
20. Rosen, H., G. Milon, and S. Gordon. 1989. Antibody to the murine type 3 complement receptor inhibits T lymphocyte-dependent recruitment of myelomonocytic cells in vivo. J. Exp. Med. 169:535.
21. Austyn, J. M., and S. Gordon. 1981. F4/80, a monoclonal antibody directed
specifically against the mouse macrophage. Eur. J. Immunol. 11:805.
22. Springer, T., G. Galfre, G. S. Secher, and C. Milstein. 1979. Mac 1: a macrophage
differentiation antigen identified by monoclonal antibody. Eur. J. Immunol.
9:301.
23. Locksley, R. M., C. B. Wilson, and S. J. Klebanoff. 1983. Increased respiratory
burst in myeloperoxidase-deficient monocytes. Blood 62:902.
24. Hsu, S. M., L. Raine, and H. Fanger. 1981. The use of avidin-biotin complex
(ABC) in immunoperoxidase techniques; a comparison between ABC and unlabelled antibody (PAP) procedures. J. Histochem. Cytochem. 29:577.
25. Rosen, H., and S. Gordon. 1990. Adoptive transfer of fluorescent labelled cells
shows that resident peritoneal macrophages are able to migrate into specialized
lymphoid organs and inflammatory sites in the mouse. Eur. J. Immunol. 20:1251.
26. McLean, I. W., and P. K. Nakane. 1974. Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. J. Histochem. Cytochem.
22:1077.
27. Ezekowitz, R. A. B., R. B. Sim, G. G. Macpherson, and S. Gordo. 1985. Interaction of human monocytes, macrophages and polymorphonuclear leucocytes
with zymosan in vitro. J. Clin. Invest. 76:2368.
28. Micklem, K. J., R. B. Sim, and E. Sim. 1984. Analysis of C3 receptor activity on
human b lymphocytes and isolation of complement receptor type 2. Biochem. J.
224:75.
29. Crocker, P. R., and S. Gordon. 1985. Isolation and characterization of resident
stromal macrophages and hematopoietic cell clusters from mouse bone marrow.
J. Exp. Med. 162:993.
30. Gailit, J., and E. Ruoslahti. 1988. Regulation of the fibronectin receptor affinity
by divalent cations. J. Biol. Chem. 263:12927.
ONTOGENY OF MURINE CR3